Biliary Mucin Research Articles

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Gallstones: The thing in itself.

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Sp702 Intraductal Papullary Mucinous Neoplasm of the Pancreas with Multi-Organ Fistulization

Background85 year old male presented with acute pancreatitis. CT imaging of the abdomen revealed severe dilation of a markedly irregular pancreatic duct with possible fistulous communication with both the stomach and duodenum. A multi-modality approach was used to achieve a diagnosis of a rare clinical entity.Endoscopic methodsUpper endoscopy defined the luminal orifaces of simultaneous pancreatico-gastric and pancreatico-duodenal fistulae. Direct pancreatoscopy was able to be performed with a 11.3 mm therapeutic videoendoscope via intubation of the pancreatico-duodenal fistula. Endoscopic ultrasound was used to image the fistulae; EUS also identified a third, pancreatico-biliary fistula. ERCP confirmed EUS findings and also allowed for extraction of biliary mucin in the clincal setting of obstructive jaundice.Clinical implicationsWe present a rare case of IPMN with simultaneous multi-organ fistulization. In this case the fistulization occurred by mechanical penetration in the absence of malignant transformation. IPMN fistulization has been described, although it is a rare clinical entity. There have been very few reported cases of multi-organ fistulization. We demonstrated a multi-modality approach to diagnosis using direct pancreatoscopy, EUS, and ERCP. Background85 year old male presented with acute pancreatitis. CT imaging of the abdomen revealed severe dilation of a markedly irregular pancreatic duct with possible fistulous communication with both the stomach and duodenum. A multi-modality approach was used to achieve a diagnosis of a rare clinical entity. 85 year old male presented with acute pancreatitis. CT imaging of the abdomen revealed severe dilation of a markedly irregular pancreatic duct with possible fistulous communication with both the stomach and duodenum. A multi-modality approach was used to achieve a diagnosis of a rare clinical entity. Endoscopic methodsUpper endoscopy defined the luminal orifaces of simultaneous pancreatico-gastric and pancreatico-duodenal fistulae. Direct pancreatoscopy was able to be performed with a 11.3 mm therapeutic videoendoscope via intubation of the pancreatico-duodenal fistula. Endoscopic ultrasound was used to image the fistulae; EUS also identified a third, pancreatico-biliary fistula. ERCP confirmed EUS findings and also allowed for extraction of biliary mucin in the clincal setting of obstructive jaundice. Upper endoscopy defined the luminal orifaces of simultaneous pancreatico-gastric and pancreatico-duodenal fistulae. Direct pancreatoscopy was able to be performed with a 11.3 mm therapeutic videoendoscope via intubation of the pancreatico-duodenal fistula. Endoscopic ultrasound was used to image the fistulae; EUS also identified a third, pancreatico-biliary fistula. ERCP confirmed EUS findings and also allowed for extraction of biliary mucin in the clincal setting of obstructive jaundice. Clinical implicationsWe present a rare case of IPMN with simultaneous multi-organ fistulization. In this case the fistulization occurred by mechanical penetration in the absence of malignant transformation. IPMN fistulization has been described, although it is a rare clinical entity. There have been very few reported cases of multi-organ fistulization. We demonstrated a multi-modality approach to diagnosis using direct pancreatoscopy, EUS, and ERCP. We present a rare case of IPMN with simultaneous multi-organ fistulization. In this case the fistulization occurred by mechanical penetration in the absence of malignant transformation. IPMN fistulization has been described, although it is a rare clinical entity. There have been very few reported cases of multi-organ fistulization. We demonstrated a multi-modality approach to diagnosis using direct pancreatoscopy, EUS, and ERCP.

Pharmacological Manipulation of Biliary Water and Lipids: Potential Consequences for Prevention of Acute Biliary Pancreatitis

Acute biliary pancreatitis, caused by macroscopic cholesterol gallstones or microlithiasis, is often a severe disease with considerable morbidity and mortality. Formation of cholesterol gallstones and microlithiasis is caused by cholesterol crystallization from cholesterol supersaturated gallbladder bile. Particularly patients with fast and extensive crystallization, due to highly concentrated bile, low biliary phospholipid contents and gallbladder mucin hypersecretion seem at risk for pancreatitis. Patients who suffered from acute biliary pancreatitis should undergo cholecystectomy as secondary prevention strategy. For patients at high surgical risk, endoscopic sphincterotomy may be an appropriate alternative. Pharmacological manipulation of biliary lipids by the hydrophilic bile salt ursodeoxycholic acid is reserved for patients with recurrent pancreatitis despite previous cholecystectomy or sphincterotomy, or with contraindications to surgical and endoscopic treatment. Maintenance therapy with ursodeoxycholic acid is however a very effective secondary prevention strategy. Potentially, secondary prevention of acute biliary pancreatitis could also be achieved through decreasing biliary mucin contents by UDCA, NSAIDs or N-acetylcystein, or through achieving bile dilution (currently not feasible).

Small Gallstones and Pancreatitis *

In our recent HEPATOLOGY article, we found that patients with biliary pancreatitis exhibit more and smaller gallbladder stones, better postprandial gallbladder emptying, and faster cholesterol crystallization than patients with symptomatic but uncomplicated gallbladder stones.1 These findings may explain in part why some gallstone patients experience pancreatitis, whereas other gallstone patients never suffer from this potentially lethal complication. We hypothesize that small gallbladder stones may migrate into the common bile duct more easily than large stones, especially in cases of preserved gallbladder emptying. Also, excess cholesterol crystals (possibly secondary to increased biliary mucin levels) could be involved: in a rat model for pancreatitis, we found that adding cholesterol crystals to hydrophobic bile salt–containing model biles infused into biliopancreatic duct markedly enhances severity of the induced pancreatitis.2 In contrast to the suggestion in the letter by Costi et al., we do not propose that “patients with asymptomatic gallstones and/or preserved motility of the gallbladder undergo prophylactic cholecystectomy.” Instead, we were careful to state that “potential benefit of prophylactic cholecystectomy in these patients remains to be elucidated.”1 Although ideally this question should be answered in a prospective randomized study, such studies will probably not be performed in the near future. In a yet unpublished study, we therefore developed a Markov model to investigate potential effects of prophylactic cholecystectomy versus “wait and see” strategy. With the aid of Monte Carlo simulations, we calculated mortality as well as number of life-years gained or lost for a 10-year period in characteristic patients with asymptomatic small (diameter < 5 mm) gallbladder stones (5,000 patients, 67% females; age 45 years). We found that prophylactic cholecystectomy may cost more life-years than treatment according to “wait and see” strategy, depending on overall pancreatitis incidence (assumed to be 0.05%, 0.1%, 0.15%, or 0.2%/year)3-5 and pancreatitis mortality (assumed to be 2.5%, 5%, 7.5%, or 10%).6-8 In 6 of 16 possible scenarios, mortality during 10 years' follow-up proved to be reduced, but only in 4 of 16 possible scenarios life-years were gained by prophylactic cholecystectomy, because surgical death occurs early, whereas gained lives occur later. Further data on incidence and subsequent mortality of pancreatitis (as well as other complications) in asymptomatic gallstone carriers are clearly needed, and further cost–benefit analyses with full-scale economic evaluations are required to determine total costs and potential benefits of prophylactic cholecystectomy versus “wait and see” strategy in the different scenarios. Niels G. Venneman*, Karel J. van Erpecum*, * Gastrointestinal Research Unit, Department of Gastroenterology and Surgery, University Medical Center Utrecht, The Netherlands.

Calcium binding to biliary mucins is dependent on sodium ion concentration: relevance to cystic fibrosis

Because hypersecretion of gallbladder (GB) mucus occurs in gallstone formation and because binding of Ca 2+ to biliary lipids only accounts for 50% of the total Ca 2+ in GB bile, we investigated the binding of Ca 2+ to human biliary mucin. Biliary mucin was purified from GB bile and binding to Ca 2+ studied. Scatchard plot analysis suggested two binding sites. Removal of sialic acid by neuraminidase resulted in 10% reduction of Ca 2+ binding, whereas, sulfatase treatment reduced Ca 2+ binding by 30%. Using a hypotonic NaCl solution, Ca 2+ binding to mucin increased curvilinearly with mucin concentration. However, binding decreased with increasing ionic strength of the NaCl solution. We conclude that binding of Ca 2+ to mucin is effected mainly through sulfate. Binding to Ca 2+ can be displaced by Na +. Ca 2+ binding to mucins is enhanced in the setting of low Na + concentrations. This phenomenon has pathophysiologic implications for the formation of thick mucus in cystic fibrosis epithelia.

Pathobiology of Cholesterol Gallstone Disease: From Equilibrium Ternary Phase Diagram to Agents Preventing Cholesterol Crystallization and Stone Formation

The primum movens in cholesterol gallstone formation is hypersecretion of hepatic cholesterol, chronic surpersaturation of bile with cholesterol and rapid precipitation of cholesterol crystals in the gallbladder from cholesterol-enriched vesicles. Associated events include biochemical defects (increased biliary mucin, and increased proportions of hydrophobic bile salts in the intestine and gallbladder), motility defects (gallbladder smooth muscle hypocontractility in vitro and gallbladder stasis in vivo, sluggish intestinal transit), and an abnormal genetic background. The study of physical-chemical factors and pathways leading to cholesterol crystallization in bile has clinical relevance and the task can be carried out in different ways. The lithogenicity of bile is investigated in artificial model biles made by three biliary lipids - cholesterol, bile salts and phospholipids - variably combined in systems plotting within the equilibrium ternary phase diagram; also, crystallization propensity of ex vivo incubated human bile is studied by biochemical analysis of precipitated crystals, polarizing quantitative light microscopy and turbidimetric methods. The present review will focus on the recent advances in the field of pathobiology of cholesterol gallstones, by underscoring the role of early events like water transport, lipid transport, crystallization phenomena - including a genetic background - in gallstone pathogenesis. Agents delaying or preventing precipitation of cholesterol crystals and gallstone formation in bile will also be discussed.

Pathobiology of Cholesterol Gallstone Disease: From Equilibrium Ternary Phase Diagram to Agents Preventing Cholesterol Crystallization and Stone Formation

The primum movens in cholesterol gallstone formation is hypersecretion of hepatic cholesterol, chronic surpersaturation of bile with cholesterol and rapid precipitation of cholesterol crystals in the gallbladder from cholesterol-enriched vesicles. Associated events include biochemical defects (increased biliary mucin, and increased proportions of hydrophobic bile salts in the intestine and gallbladder), motility defects (gallbladder smooth muscle hypocontractility in vitro and gallbladder stasis in vivo, sluggish intestinal transit), and an abnormal genetic background. The study of physical-chemical factors and pathways leading to cholesterol crystallization in bile has clinical relevance and the task can be carried out in different ways. The lithogenicity of bile is investigated in artificial model biles made by three biliary lipids - cholesterol, bile salts and phospholipids - variably combined in systems plotting within the equilibrium ternary phase diagram; also, crystallization propensity of ex vivo incubated human bile is studied by biochemical analysis of precipitated crystals, polarizing quantitative light microscopy and turbidimetric methods. The present review will focus on the recent advances in the field of pathobiology of cholesterol gallstones, by underscoring the role of early events like water transport, lipid transport, crystallization phenomena - including a genetic background - in gallstone pathogenesis. Agents delaying or preventing precipitation of cholesterol crystals and gallstone formation in bile will also be discussed.

Lith genes control mucin accumulation, cholesterol crystallization, and gallstone formation in A/J and AKR/J inbred mice

We recently identified 2 Lith genes that determine cholesterol gallstone formation in C57L/J inbred mice, which show a gallstone prevalence of ∼80% on feeding 1.0% cholesterol and 0.5% cholic acid. The aim of this study was to explore if the same Lith loci contribute to the variation in gallstone susceptibility in a new experimental cross. After 12 weeks of feeding the lithogenic diet to inbred mice of strains A/J and AKR/J as well as their F 1 progeny, we used microscopy of bile to assess mucin accumulation, crystallization pathways, and stone formation. Backcross progeny (n = 225) were phenotyped and genotyped selectively for microsatellite markers spanning the genome. Quantitative trait loci (QTL) affecting gallstone phenotypes were identified by linkage analysis. Both inbred strains showed accumulation of mucin gel and cholesterol supersaturation. However, only strain AKR developed gallstones (prevalence of 20%), whereas strain A showed a stable liquid crystalline state and no stones. QTL analysis identified a gallstone locus on chromosome 17 ( Lith3). A second gene locus on chromosome 15 that controls mucin accumulation harbors the mucin gene Glycam1, which was shown to be expressed in gallbladder epithelia by immunohistochemistry. Gallstone and mucin loci colocalized with potential QTLs affecting the formation of cholesterol crystals. In conclusion, QTL analysis identified specific gene loci determining mucin accumulation, cholesterol crystallization, and gallstone formation. Characterization of the pathophysiologic roles of Lith3 and the new biliary mucin gene Glycam1 might provide insights into primary defects of human cholelithiasis and lead to new therapeutic strategies for prestone intervention. (H EPATOLOGY 2002;36:1145-1154.)

Cystic fibrosis transmembrane conductance regulators (CFTR) in biliary epithelium of patients with hepatolithiasis.

Cystic fibrosis transmembrane conductance regulator (CFTR) has been considered to be involved in the regulatory pathway of biliary mucin secretion. We investigated expression of CFTR protein and mRNA in 24 livers with hepatolithiasis, in 6 with cholangiocarcinoma, and in 12 histologically normal livers. According to the histologic features of chronic proliferative cholangitis, hepatolithiasis was subdivided into inflammatory cell infiltration predominant (N = 14) and fibrosis predominant (N = 10). The mean signal density of CFTR in overall hepatolithiasis and in histologically normal livers was 1.23 +/- 0.15 and 1.01 +/- 0.13, respectively (P > 0.05). The CFTR protein (1.60 +/- 0.18) and mRNA (1.09 +/- 0.15) in inflammatory cell infiltration predominant patients were significantly higher (CFTR protein, 1.01 +/- 0.13; mRNA, 0.75 +/- 0.11) than in control subjects (P < 0.05), whereas those in fibrosis-predominant patients (CFTR protein, 0.72 +/- 0.15; mRNA, 0.55 +/- 0.13) were less than in control subjects (P < 0.05). CFTR protein (1.27 +/- 0.17) in patients with cholangiocarcinoma was not different from that of control subjects (1.01 +/- 0.13). CFTR expression in hepatolithiasis patients was rather heterogeneous but was closely related to the histology of bile ducts.

Human biliary mucin binds to E-selectin: a possible role in modulation of inflammation

E-selectin, expressed on endothelial cells, mediates adhesion of leukocytes and tumor cells to endothelium. CA19-9 (sialyl-Lewis(a)) and sialyl-Lewis(x) are specific ligands for E-selectin. We have recently shown that mucin-rich culture media from human gallbladder epithelial cells contains CA19-9. In this study, we have tested whether human biliary mucin binds to E-selectin. The ability of mucins to inhibit the adhesion of HL-60 cells to immobilized E-selectin was taken as an index for E-selectin binding. Gallbladder bile, hepatic bile, and culture medium from human gallbladder epithelial cells completely inhibited the adhesion of HL-60 cells to E-selectin. The mucin-rich fractions of human bile exhibited strong inhibition, whereas mucin-free fractions had little effect. In contrast to human bile samples, CA19-9-free medium from cultured dog gallbladder epithelial cells failed to inhibit HL-60 binding. Furthermore, after CA19-9 immunoaffinity chromatography, which selectively extracted CA19-9 from bile, bile samples showed poor inhibition of HL-60 adhesion to immobilized E-selectin. A good correlation was observed between E-selectin binding and CA 19-9 concentrations in bile. Our results show that human bile has E-selectin binding activity that is mediated by the CA19-9 side chain of biliary mucin.

Secretory low-molecular-weight phospholipases A2 and their specific receptor in bile ducts of patients with intrahepatic calculi: factors of chronic proliferative cholangitis.

Intrahepatic calculi is characterized by an intractable course and frequent recurrences, requiring multiple operative interventions. Chronic proliferative cholangitis, an active and long-standing inflammation of the stone-containing bile ducts with the hyperplasia of epithelia and the proliferation of the duct-associated mucus glands, may underlie the complex nature of the disease. In terms of the pathophysiology, interest has been focused on the role of secretory low-molecular-weight phospholipases A2 (sPLA2s) as inflammatory mediators or factors modulating cell functions via their specific sPLA2-receptor, and also on the production and secretion of altered mucin molecules from the inflamed bile ducts. In search of factors involving chronic proliferative cholangitis, the sPLA2 isoforms in the bile such as the pancreatic-type sPLA2 (group IB sPLA2) and the arthritic-type sPLA2 (group IIA sPLA2), were assayed to correlate protein masses of the sPLA2s with alterations in biliary composition. Furthermore, the steady-state messenger RNA (mRNA) levels of the sPLA2s, the membrane-bound sPLA2-receptor, cystic fibrosis transmembrane conductance regulator (CFTR), and mucin core polypeptide (MUC) genes in the bile ducts were assayed by reverse- transcriptase polymerase chain reaction (RT-PCR). Immunoreactive sPLA2-IB and sPLA2-IIA levels were significantly higher in the bile from the stone-containing hepatic ducts (2315 +/- 677 for sPLA2-IB; 281 +/- 42 for sPLA2-IIA ng/dL, mean +/- SEM; n = 20) than in the ductal bile from gallbladder stone patients (609 +/- 92, P <.01; 22 +/- 2, P <.01; n = 24). The increased sPLA2 levels were associated with a concomitant increase in lysophosphatidylcholine, prostaglandin E2 (PGE2), and total mucin concentrations. The affected bile ducts showed an increased mRNA level of sPLA2-IB and sPLA2-IIA compared with the ducts from control subjects, in whom the mRNAs of the sPLA2-receptor and other sPLA2 isoforms, such as groups V and X sPLA2s, were coincidently expressed. Reflecting the increased amounts of total biliary mucins, the affected ducts showed an increase in mRNA levels of CFTR as well as MUC2, MUC3, MUC5AC, MUC5B, and MUC6 compared with the ducts from control subjects. In intrahepatic calculi, an enhanced expression of the sPLA2s and their possible cross-talk via sPLA2-receptor may be of pathophysiological significance for the chronic proliferative cholangitis, in association with the enhanced CFTR expression and the alterations in mucin gene expression in the bile ducts, probably through potentiating arachidonate metabolism with associated biliary alterations favoring growth of preexisting stones and even further progressions.

Lipid peroxidation in bile: the role of hydrophobic bile acids and the effect on biliary epithelial cell function

Reactive oxygen species and lipid peroxidation have been implicated in a wide variety of pathophysiological conditions. Persistent oxidant stress in plasma and tissue samples has been demonstrated in various hepatobiliary diseases. Oxidant stress may cause peroxidation of biliary lipids. In the presence of transition metal ions, hydrophobic bile acids cause an increase in lipid peroxidation. Biliary lipid peroxidation may affect the function of the biliary epithelium. Activation of epithelial nuclear factor kappa-B (NF- κB), which is known to up-regulate several pro-inflammatory genes, appears to mediate the latter process. Mucin secretion is one of the important functions of the biliary epithelium. Lipid peroxidation induces mucin hypersecretion by biliary epithelial cells. Biliary mucins are of pathophysiological relevance in cholesterol gallstone disease and other hepatobiliary diseases. This review aims to discuss the link between hydrophobic bile acids, biliary lipid peroxidation and biliary epithelial cell function.

Mucins and calcium phosphate precipitates additively stimulate cholesterol crystallization

Human biliary mucin and calcium binding protein (CBP) influence formation of both calcium salt precipitates and cholesterol crystals and colocalize in the center of cholesterol gallstones. We investigated how physiological concentrations of these proteins regulate cholesterol crystallization in model biles, supersaturated with cholesterol and calcium salts, mimicking pathological human bile. Using polarizing light microscopy and nephelometry to assess cholesterol crystallization, the influence of calcium ions and calcium phosphate precipitates in the absence and presence of mucin, CBP, and human serum albumin was determined. Calcium phosphate precipitates stimulated cholesterol crystallization more strongly than soluble calcium. Mucin also stimulated, and with soluble calcium or calcium phosphate precipitates additively increased, the cholesterol crystal mass. In the absence of mucin, only human serum albumin plus CBP, not these proteins individually, decreased the stimulating effect of calcium phosphate precipitates but not of soluble calcium. However, seeding of calcium phosphate precipitates in biles with mucins resulted in near complete cholesterol crystallization within one day whether CBP and HSA were or were not also present.▪ In conclusion, calcium salt precipitates plus human biliary mucins induce rapid and complete cr ystallization of cholesterol from model biles, little influenced by human biliary calcium binding proteins.—van den Berg, A. A., J. D. van Buul, G. N. J. Tytgat, A. K. Groen, and J. D. Ostrow. Mucins and calcium phosphate precipitates additively stimulate cholesterol crystallization.

Human biliary mucin binds to E-selectin: A possible role in modulation of inflammation and tumor metastasis

Human biliary mucin binds to E-selectin: A possible role in modulation of inflammation and tumor metastasis

Regulation of mucin secretion in human gallbladder epithelial cells: Predominant role of calcium and protein kinase C

BACKGROUND & AIMS: The cellular mechanisms that regulate biliary mucin secretion in humans are unknown. To address this question, human gallbladder epithelial cells were used in primary culture.METHODS: [1- (14)C]-glucosamine-labeled glycoproteins secreted in vitro were analyzed and quantified after exposing cells to activators and inhibitors of the main transduction pathways and to potential biologically active secretagogues.RESULTS: Secreted glycoproteins showed characteristics of biliary mucins. Activators of adenosine 3',5'- cyclic monophosphate-dependent pathway as well as secretin and vasoactive intestinal polypeptide did not significantly modify mucin secretion. By contrast, ionomycin and phorbol-12-myristate 13-acetate increased mucin secretion by 292% +/- 48% and 134% +/- 19% over basal level, respectively. The effects of these two agents were additive and were mediated by a calcium-dependent pathway implicating Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) and by the activation of protein kinase C (PKC), respectively, as ascertained by using inhibitors. Mucin secretion was stimulated by extracellular adenosine 5'-triphosphate via P2U receptors, cytosolic calcium increase, and PKC and by taurochenodeoxycholate via cytosolic calcium increase and Ca2+/CaM-kinase II.CONCLUSIONS: Mucin secretion in human gallbladder is regulated predominantly by calcium-dependent pathways implicating Ca2+/CaM-kinase II and PKC. Extracellular adenosine 5'- triphosphate and taurochenodeoxycholate may play a role in the regulation of biliary mucin secretion by activating these different signaling pathways.(Gastroenterology 1997 Mar;112(3):978-90)

Quantification of mucin in human gallbladder bile: a fast, specific, and reproducible method

It is generally accepted that gallbladder mucin (GBM) plays an important role in the pathogenesis of cholesterol gallstone (ChG) disease. However, it remains unclear whether ChG patients have higher GBM concentrations than controls. Discrepant findings regarding biliary mucin concentrations may be due to methodological problems with the assays commonly used. The methods currently used to quantitate mucin in bile have not been systematically evaluated. To establish a reliable method for mucin quantification in bile, we evaluated three mucin assays: the classic Pearson-PAS (periodic acid Schiff) assay, a direct fluorometric assay, and a new PAS or fluorometric assay with the following modifications of the Pearson assay: preincubation of bile samples with TBS containing KSCN and sodium taurocholate and micellation of biliary lipids during gel chromatographic fractionation using 25 mM sodium taurocholate in the elution buffer. SDS-PAGE and monoclonal anti-human-GBM (GBM59) were used to identify mucin. Highly specific and reproducible mucin isolation was achieved with the modified method. We found considerable loss of mucin during the different purification steps in the Pearson method. The direct fluorometric assay showed unspecific fluorometric signal with low molecular constituents of bile. Our experiments showed that human-GBM can be accurately measured after a simple modified chromatographic fractionation followed by a PAS or fluorometric assay.

Biliary mucin secreted by cultured human gallbladder epithelial cells carries the epitope of CA19-9: [formula omitted], M. I. Eder, P. Stieber∗∗, G. Meyer∗, C. Reuter∗, D. Jüngst, R. Lamerz, G. Paumgartner, Depts of Medicine, II, Surgery∗, and Clin. Chemistry∗∗, Klinikum Grosshadern, Ludwig-Maximilians-University, 81366 Munich, Germany

Serum CA 19-9 is increased in patients with different gastrointestinal malignancies. Unfortunately, CA 19-9 is also detected in high concentrations in normal bile causing unspecific serum elevations during inflammatory disease of the biliary tract and cholestasis. In order to identify the source of CA 19-9 in bile, the capacity of cultured human gallbladder epithelial cells (HGBEC) to secrete CA 19-9 was investigated. Cells were harvested from gallbladders removed by cholecystectomy and cultured for up to 14 days in collagen I coated 24-well culture dishes. CA 19-9 was measured in the culture medium by a solid-phase CA 19-9 EIA (Boehringer). In addition, culture medium was separated by Sepharose 4B-Cl, Concanavalin-A (Con-A) and CA 19-9 affinity chromatography. Significant CA 19-9 activity was measured in the culture medium after a 24 hour incubation period. Following separation of the culture medium by Sepharose 4B-Cl and Con-A affinity chromatography, 90% of the CA 19-9 activity was recovered in the exclusion volume (> 2000 kDa) from which 90% were identified as Con-A negative. A close correlation was found between CA 19-9 and concentrations of mucin purified from human gallbladder bile. Furthermore, CA 19-9 affinity chromatography selectively extracted mucins from the culture medium of HGBEC. Finally, addition of the mucin secretagogue bethanechol (6 mM) to the culture medium increased CA 19-9 activity in the medium. In conclusion, normal HGBEC secrete mucins carrying the epitope of CA 19-9. During inflammatory biliary disease unspecific elevation of CA 19-9 in serum may reflect both inflammatory hypersecretion and leakage of biliary mucins into serum.

Fibronectin in human gallbladder bile: cholesterol pronucleating and/or mucin "link" protein?

Some biliary proteins (pronucleators) seem to be essential factors for cholesterol crystal formation and crystal growth in bile. A recent study suggests that fibronectin is such a pronucleator in bile. Fibronectin also seems to closely interact with intestinal mucin. Since biliary mucin plays an important role in gallstone formation, such an interaction in bile may be of relevance in cholesterol gallstone formation. To more clearly elucidate the role of fibronectin in cholesterol gallstone disease, we measured the concentration of fibronectin in native bile of cholesterol gallstone patients and checked its influence on the cholesterol nucleation time of model bile. We further looked for a molecular interaction between biliary fibronectin and gallbladder mucin. We found that fibronectin is present in gallbladder bile of gallstone patients in low concentrations (2.6 +/- 1.2 micrograms/ml). Bile fibronectin did not interact with gallbladder mucin. Moreover, in a wide range of concentrations fibronectin had no influence on the nucleation time of model bile. We conclude that fibronectin does not seem to play a major role in cholesterol gallstone disease.

Heterogeneity of Human Biliary Mucin: Functional Implications

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67-74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained alpha-D-N-acetylglucosamine and were fucosylated, sialylated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins.(ABSTRACT TRUNCATED AT 250 WORDS)